Black cohosh is an increasingly popular alternative to estrogen replacement therapy for the relief of menopausal symptoms, primarily hot flushes. However, an important consideration for long-term therapy is potential toxicity and carcinogenicity. Therefore, we undertook a study to assess the estrogenic activity of black cohosh to examine its safety for those with, or at high risk of developing, breast cancer. Several assays were utilized as listed: RNAse protection assays, which ascertain the regulation of the expression of E2-responsive genes; estrogen-responsive-element (ERE)-luciferase, which determines modulation of the ER function by transactivation of the ERE; the Ishikawa cell system, which has an E2-regulated endogenous alkaline phosphatase; and colony formation of ER-expressing breast cancer cells, which indicates possible progression of early stage breast cancer into a more aggressive state. Black cohosh extracts did not demonstrate estrogenic activity in any of these assay systems. This is an encouraging step in the assessment of the safety of black cohosh for treatment of menopausal hot flushes.